Beta-Amyloid precursor protein (BetaAPP) is a highly conserved and ubiquitously expressed integral membrane protein encoded in humans on chromosome 21. During aging in primates and certain other mammals, and particularly in humans with Alzheimer's disease or trisomy 21, the amyloid Beta-protein (BetaA4) is progressively deposited in cerebral plaques and meningocerebral blood vessels. The Beta-amyloid precursor gene produces a number of alternatively spliced transcripts. The most widely studied transcripts encode proteins of 695, 751 and 770 amino acids. BetaAPP 751 and BetaAPP 770 both contain a region of 56 amino acids with high homology to the Kunitz family of protease inhibitors. Both platelets and endothelial cells contain membrane-inserted BetaAPP and release the soluble extramembranous portion of the protein, protease nexin 2 (PN 2), into the extracellular environment following constitutive proteolytic cleavage at residue 687 of BetaAPP. PN 2 is stored in the alpha-granules of platelets and released upon platelet activation. PN 2 has been demonstrated to be a potent inhibitor of factor XIa and to promote cell adhesion. We have recently defined distinct patterns of membrane-associated and soluble BetaAPP isoforms in platelets and lymphocytes and defined a second constitutive processing pathway for BetaAPP in human endothelial cells involving reinternalization of cell surface BetaAPP and its trafficking to lysosomes where an array of carboxyl terminal fragments containing BetaAPP are generated. In view of the relevance of this potentially amylodogenic pathway to the microvascular deposition of BetaA4 and the probable role of platelet and endothelial cell BetaAPP in hemostasis and thrombosis we propose to study posttranslational processing, subcellular localization of BetaAPP in resting and activated platelets and the function of BetaAPP in cellular and mouse models. Characterization of the ultrastructural localization and dynamic cellular processing of BetaAPP in platelets will be studied with particular attention to the relative utilization by BetaAPP of the alpha-granule secretory pathway (releasing PN2), alpha-granule membrane insertion, trafficking to lysosomes and generation of amyoloidogenic peptides. These processes will be studied by use of immunogold electron microscopy, biotinylation or antibody labeling of surface BetaAPP, and by direct purification of lysosomes and alpha-granules from platelets and biochemical analysis of mature BetaAPP and BetaA4-containing fragments. The processing and subcellular localization in normal cells will be compared to that in platelets from subjects with familial Alzheimer's disease and trisomy 21. To determine the cellular function of BetaAPP we will use homologous recombination to eliminate or alter one copy or both copies of the BetaAPP gene in murine embryonic stem cells (ES), AtT20 cells (which have storage granules), and 293 cells (which do not contain storage granules). The phenotype of these cells will be characterized with respect to viability, PN2-like activity expressed on the cell surface or secreted, and with regard to localization and processing of BetaAPP forms. The hemizygous or heterozygous ES cells will be used to generate mice deficient in BetaAPP or expressing mutant BetaAPP.